As the Human Genome Project and other genome sequencing efforts are completed, the demand for various Deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and protein has been increased in a great deal from life science researchers. For example, RNA extraction is required to compare differentially expressed gene between normal tissues and tumor tissues, or between tumor cells and drug treated tumor cells. Also, human genome profiling and gene expression profiling require the comparison of DNA, RNA and protein from the same piece of biomaterials. Extraction of high quality DNA, RNA and protein simultaneously without cross contamination are very difficult, especially when the biological samples are very limited. For example, amounts of clinical specimens are so small that it is impossible for life science researchers to divide them into even smaller sections to extract DNA, RNA and protein separately.
There is no appropriate or dependable technique for isolation of DNA, RNA and protein simultaneously from biological samples. Cesium chloride (CsCl) gradient is a traditional method for extracting DNA and RNA at the same time, but no protein is extracted. Very few people are using this method for RNA and DNA extraction because of its time consuming and high expense. Column purification of RNA and DNA from tissue without protein is another method but the yield is relative low and some populations of RNA and DNA are lost.
A commercial kit method claims that it can extract DNA, RNA and protein from biomaterials at the same time with acidic phenol or acidic extraction (low pH) reagents (U.S. Pat. No. 5,346,994). However, the major application of this method is designed to extract RNA only because the quality of DNA and protein is so poor that they cannot be used in any real application as DNA and protein are intended to be. In this methods DNA is moved into organic phase of extraction by reagents with low pH (or acidic extraction reagents) in order to separate DNA and RNA, while RNA stays in aqueous phase. But this kit method has to pay tremendous sacrifices for losing the populations of RNA with large size as these populations of RNA moved into organic phase along with DNA, thereby causing substantial contamination to DNA due to the presence of protein or unknown materials to be used, such that the entirety of protein population is lost so much that protein cannot be used in almost any applications. The procedures of this kit method with acidic extraction reagents are described briefly as follows: biomaterials are homogenized in these acidic extraction reagents, in addition of chloroform extraction followed by centrifugation, the homogenate will be separated in three phases: aqueous phase, inter phase and organic phase. RNA stays in the aqueous phase, DNA and protein are in the inter phase and organic phase. Further process on inter phase and organic phase can separate DNA and protein. Then DNA, RNA and protein can be isolated respectively.
Although the kit method with acidic extraction reagent gives RNA with acceptable quality from cell lines, the quality of DNA, RNA and protein extracted from biomaterials, especially primary tissues, are very compromised compared with the other conventional extraction methods, such as CsCl gradient centrifugation, protease K digestion for DNA extraction, and non-ionic detergent for protein extraction. loosing large molecules of DNA, RNA and protein and decreasing recovery rate of DNA and protein DNA and protein indicate that some population of DNA, RNA and protein are lost from the extraction by reagents with low pH used in commercial kit. The lacking of stability, purity and some populations of the DNA, RNA and protein extracted by reagents with low pH as used in kit method, especially from tissues, are serious problems. One of such example is when these DNA, RNA and protein are used in gene expression profiling that becomes more and more demanding in terms of completed and representative populations of DNA, RNA and protein. Other major drawback to prevent users from using this kit method to extract DNA, RNA and protein simultaneously is that processes are too complicated to carry on, especially to extract DNA and protein.
Poor qualities of DNA, RNA and protein extracted by reagents with low pH as used in kit method are caused by several reasons. First, pre-mixture of denaturing reagents such as Guanidine and phenol can lead to incomplete denaturing RNase existing in biomaterials, which degrades RNA during processes afterward. Second, extraction by reagents with low pH moves only a portion of genomic DNA into organic phase, which causes lower yield of DNA in later extraction; furthermore, extraction by reagents with low pH also moves some populations of RNA with extra large molecular weight (size) into organic phase, which reduces the yield of RNA population with extra large molecular weight (size). Third, DNA and protein may tangle together each other in organic phase to prevent DNA and protein from further separation. For instance, separation of DNA and protein in organic phase with ethanol could precipitate and lose protein with extra large molecular weight (size) and certain populations of protein (fragment of membranes) in pellet of DNA; in addition, DNA could not be precipitated completely from organic phase with lower concentration of ethanol, which decreases the yield of DNA. Fourth, isolations of DNA and protein involve many complicated steps with harsh conditions, which could break down the molecules of DNA and protein. Any one or any combination of four reasons described above will badly compromise the qualities of DNA, RNA and protein extracted by reagents with low pH as used in kit method.